US8618506B2 - Fluorescence life measuring apparatus, fluorescence life measuring method and program - Google Patents

Fluorescence life measuring apparatus, fluorescence life measuring method and program Download PDF

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Publication number
US8618506B2
US8618506B2 US13/143,518 US201013143518A US8618506B2 US 8618506 B2 US8618506 B2 US 8618506B2 US 201013143518 A US201013143518 A US 201013143518A US 8618506 B2 US8618506 B2 US 8618506B2
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fluorescence
afterglow
fluorescence life
stage
fluorescent material
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US20110266458A1 (en
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Isamu Nakao
Koshi Tamamura
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Sony Corp
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Sony Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Definitions

  • the present invention relates to a fluorescence life measuring apparatus, a fluorescence life measuring method and a program, which is useful in a field in which a technique of detecting the life of emitted light caused by irradiation of excitation light is used.
  • a fluorescence life measuring apparatus irradiates a specimen supported in a cell with pulsed excitation light, measures the time waveform of emitted fluorescence caused by the excitation light using a photomultiplier tube or streak camera, and obtains the fluorescence life from the measurement result (for example, see Patent Document 1).
  • the photomultiplier tube or streak camera is relatively large itself, which makes such a fluorescence life measuring apparatus large as a whole. Also, in this fluorescence life measuring apparatus, a light source for irradiating with pulsed excitation light is used, and the photomultiplier tube or streak camera needs to be driven according to the timing of irradiation from the light source, which requires complicated and time-consuming adjustment process of the photomultiplier tube or streak camera.
  • the invention proposes a fluorescence life measuring apparatus, a fluorescence life measuring method and a program that can obtain fluorescence life using a simple configuration.
  • the invention provides a fluorescence life measuring apparatus including: a moving means for moving a stage on which a fluorescent material to be measured is placed; an irradiating means for irradiating with excitation light the fluorescent material placed on the stage moved at a constant speed by the moving means; an imaging means for imaging afterglow of emitted fluorescence caused by the excitation light; and a fluorescence life calculating means for using an image imaged by the imaging unit to detect the elapsed time from a fluorescence position and afterglow strength at a target afterglow position and calculate the fluorescence life.
  • the invention provides a fluorescence life measuring method including: a moving control step of moving a stage on which a fluorescent material to be measured is placed; an irradiating step of irradiating with excitation light the fluorescent material placed on the stage moved at a constant speed through the control in the moving control step; an imaging step of imaging afterglow of emitted fluorescence caused by the excitation light; and a fluorescence life calculating step of using an image imaged in the imaging step to detect the elapsed time from a fluorescence position and afterglow strength at a target afterglow position and calculate the fluorescence life.
  • the invention provides a program for causing: a moving control means to move a stage on which a fluorescent material to be measured is placed; an irradiating means to irradiate with excitation light the fluorescent material placed on the stage moved at a constant speed by the moving control means; an imaging means to image afterglow of emitted fluorescence caused by the excitation light; and a calculating unit to use an image imaged by the imaging means to detect the elapsed time from a fluorescence position and afterglow strength at a target afterglow position and calculate the fluorescence life.
  • the fluorescence life measuring apparatus captures the fluorescence life in the form of an afterglow image in the moving direction, which allows obtaining of the strength change of light occurring in a short period with a common camera rather than a photomultiplier tube or streak camera, resulting in a simple configuration of the fluorescence life.
  • FIG. 1 schematically shows a configuration of a fluorescence life measuring apparatus.
  • FIG. 2 schematically shows a structure of a disc stage.
  • FIG. 3 shows a configuration example of an optical system.
  • FIG. 4 schematically shows a beam shape on a light receiving surface.
  • FIG. 5 is a block diagram showing a configuration of a measuring unit.
  • FIG. 6 is a photograph of an imaging result when excitation light is irradiated.
  • FIG. 7 is a graph showing afterglow strength change over time.
  • FIG. 8 is a flowchart showing a fluorescence life measuring process.
  • FIG. 1 shows a schematic configuration of a fluorescence life measuring apparatus 1 .
  • the fluorescence life measuring apparatus 1 includes a disc-like stage (hereinafter referred to as “disc stage”) 2 , a laser light source 3 , an optical system 4 , a focus control unit 5 , an imaging unit 6 and a measuring unit 7 .
  • the disc stage 2 is detachably supported on a rotating shaft SA through a through hole provided at the center of the disc stage 2 .
  • FIG. 2 shows a structure of the disc stage 2 .
  • the disc stage 2 has a layer structure in which a reflection film 2 B and a fluorescence film 2 C are formed in this order on one surface of a disc substrate 2 A.
  • the disc substrate 2 A is formed of a material that becomes transparent when irradiated with light to excite the fluorescence film 2 C (hereinafter referred to as “excitation light”) and has a thickness of, for example, about 1.3 mm. Specifically, quartz is used as the material, for example.
  • the reflection film 2 B is formed of a material that can cause a predetermined amount of interface reflection and has a uniform thickness of, for example, about 100 nm. Specifically, titanium oxide is used as the material, for example, which causes about 20% of interface reflection when the excitation light has a wavelength of 405 nm and the disc substrate 2 A is formed of quartz.
  • the fluorescence film 2 C is formed of an organic or inorganic material as a target of fluorescence life measurement and has a uniform thickness of, for example, about 100 nm.
  • the laser light source 3 is positioned facing the surface of the disc substrate 2 A other than the surface on which the reflection film 2 B is formed and is configured to irradiate the fluorescence film 2 C with the excitation light.
  • the laser light source 3 used in this embodiment irradiates with the excitation light having a wavelength of 405 nm, an output power of 1 mW and a lateral mode of TEM00.
  • the optical system 4 is configured to collect the excitation light irradiated by the laser light source 3 onto the interface between the disc substrate 2 A and the reflection film 2 B and guide the light reflected from the interface to the focus control unit 5 .
  • FIG. 3 shows a specific configuration example of the optical system 4 .
  • laser light emitted from the laser light source 3 as linearly polarized light parallel to the plane of paper is converted into parallel light by a collimator lens 11 and guided to a polarizing beam splitter 12 . Then, in the optical system 4 , laser light having passed through the polarizing beam splitter 12 is converted to circularly polarized light by a quarter wavelength plate 13 and collected onto the interface between the disc substrate 2 A and the reflection film 2 B by an objective lens 14 .
  • the fluorescent material to be measured formed as the fluorescence film 2 C, is excited to emit fluorescence spatially isotropically.
  • the optical system 4 light reflected from the interface between the disc substrate 2 A and the reflection film 2 B is guided through the objective lens 14 to the quarter wavelength plate 13 and converted to s-polarized light by the quarter wavelength plate 13 . Then, in the optical system 4 , the reflected light converted to s-polarized light is reflected by 90° from the polarizing beam splitter 12 and guided through a condensing lens 15 and a cylindrical lens 16 to the focus control unit 5 ( FIG. 1 ).
  • the focus control unit 5 includes a light receiving unit for receiving light guided from the optical system 4 .
  • FIG. 4 shows the shape of light guided from the optical system 4 on the light receiving surface of the light receiving unit.
  • the shape of light is circular as shown by broken line.
  • the shape of light is elliptical as shown by alternate long and short dash line, due to aberration occurring in the cylindrical lens 16 .
  • the long axis and short axis of the elliptical shape alternate depending on whether the focus is short of or beyond the interface between the disc substrate 2 A and the reflection film 2 B.
  • the focus control unit 5 generates focus control signal from the signal obtained from the light receiving unit as a result of photoelectric conversion in each of divided four regions. Specifically, in the example shown in FIG. 4 , signals of the regions A to D are obtained, then (A+D) ⁇ (B+C) is calculated to generate the focus control signal.
  • the focus control unit 5 controls an actuator that can move in the optical axis direction according to the focus control signal to move the objective lens 14 ( FIG. 3 ) provided on the actuator so that the focus is positioned on the interface between the disc substrate 2 A and the reflection film 2 B.
  • the imaging unit 6 is provided opposite the fluorescence film 2 C of the disc stage 2 and is configured to image fluorescence emitted from the fluorescence film 2 C and give the imaging result to the measuring unit 7 as imaging data.
  • the measuring unit 7 is configured to calculate fluorescence life based on the imaging data given by the imaging unit 6 .
  • FIG. 5 shows a schematic configuration of the measuring unit 7 .
  • the measuring unit 7 is configured such that various hardware devices are connected to a CPU (Central Processing Unit) 21 via a bus 22 .
  • CPU Central Processing Unit
  • the measuring unit 7 includes, as the hardware devices, at least a ROM (Read Only Memory) 23 , a RAM (Random Access Memory) 24 as work memory of the CPU 21 and an interface 25 .
  • the measuring unit 7 also includes an input device 26 for inputting an instruction according to a user operation, a display 27 and a storage 28 .
  • the ROM 23 stores a program for performing fluorescence life measuring process (hereinafter referred to as “fluorescence life measuring program”).
  • the interface 25 is connected to a spindle motor SM attached to the rotating shaft SA ( FIG. 1 ), the laser light source 3 , the focus control unit 5 and the imaging unit 6 ( FIG. 1 ).
  • the CPU 21 expands in the RAM 24 the fluorescence life measuring program stored in the ROM 23 and functions as a driver 31 , a preprocessor 32 and a fluorescence life calculator 33 .
  • the driver 31 drives the laser light source 3 to irradiate with the excitation light in a predetermined period and drives the focus control unit 5 so that the focus is positioned on the interface between the disc substrate 2 A and the reflection film 2 B. Further, the driver 31 drives the spindle motor SM to rotate at a constant linear speed and drives the imaging unit 6 to image in a predetermined period.
  • FIG. 6 shows an imaging result of momentarily irradiating with the excitation light a predetermined position on the interface between the fluorescence disc substrate 2 A and the reflection film 2 B when the rotating disc stage 2 is at a position to be irradiated.
  • a 14-bit digital still camera is used as the imaging unit 6 , which includes a lens with a focal length of 6.33 to 19 mm and an 8.1 million pixel interlaced 1/2.5-inch CCD (Charge Coupled Device).
  • CCD Charge Coupled Device
  • the preprocessor 32 calculates the ratio of the strength at the tail position (the black circle area farthermost from the irradiated position in FIG. 6 ) to that at the head position (the black circle area nearest to the irradiated position in FIG. 6 ) of the afterglow of the fluorescence emitted at the irradiated position in an image represented by the imaging data given by the imaging unit 6 .
  • the preprocessor 32 adjusts the rotation speed of the disc stage 2 or the angle of view of the imaging unit 6 through the interface 25 so that the ratio falls within a defined range. Consequently, the afterglow from the irradiated position to a position at which a target strength is maintained (for example, a position just before disappearance) falls within the imaging range.
  • the fluorescence life calculator 33 calculates the strength and elapsed time of the afterglow from the head position to the tail position at predetermined intervals (at black circles in FIG. 6 ) and creates a table showing the change of the afterglow over time (hereinafter referred to as “afterglow change table”) based on the calculation result.
  • FIG. 7 shows the afterglow change table for the afterglow shown in FIG. 6 .
  • the elapsed time can be determined from the position information of the afterglow and the radius and rotation speed of the disc stage.
  • the fluorescence film 2 C ( FIG. 2 ) formed on the disc stage 2 is not limited to have a single excitation state, but may have multiple excitation states.
  • the afterglow change table does not show a linear relationship as shown in FIG. 7 , but shows a mixture of multiple linear relationships depending on the number of the excitation states.
  • the fluorescence life calculator 33 can calculate the fluorescence life of the fluorescence film 2 C having multiple excitation states by designating linear portions having different gradients and curved portions of the slope in the afterglow change table as positions to be noted.
  • the fluorescence life is calculated to be 1.8 msec when the linear speed of the disc stage 2 in imaging shown in FIG. 5 is 1 m/sec.
  • the CPU 21 When an instruction to measure the fluorescence life is given, the CPU 21 starts the fluorescence life measuring process to proceed to a first step SP 1 .
  • the CPU 21 drives the spindle motor SM to rotate the disc stage 2 at a constant linear speed, then proceeds to a second step SP 2 .
  • the CPU 21 drives the laser light source 3 to irradiate the disc stage 2 with the excitation light in a predetermined period, then proceeds to a third step SP 3 .
  • the CPU 21 drives the focus control unit 4 to position the focus on the interface between the disc substrate 2 A and the reflection film 2 B, then proceeds to a fourth step SP 4 .
  • the CPU 21 drives the imaging unit 6 to start capturing an image at the time of irradiating the disc stage 2 with the excitation light, then proceeds to a fifth step SP 5 .
  • the CPU 21 causes the afterglow from the irradiated position to a position at which a target strength is maintained (for example, a position just before disappearance) of the image captured in the fourth step SP 4 to fall within the imaging range, then proceeds to a sixth step SP 6 .
  • a target strength for example, a position just before disappearance
  • the rotation speed of the disc stage 2 or the angle of view of the imaging unit 6 is adjusted so that the ratio of the strength at the tail position to that at the head position of the afterglow of the fluorescence emitted at the irradiated position falls within a defined range.
  • the CPU 21 calculates the strength and elapsed time of the afterglow from the head position to the tail position at predetermined intervals, then proceeds to a seventh step SP 7 .
  • the CPU 21 creates an afterglow change table based on the calculation result of the sixth step SP 6 , then proceeds to an eighth step SP 8 .
  • the CPU 21 calculates the fluorescence life based on the equation (1), then ends the fluorescence life measuring process. Note that the CPU 21 is configured so that, when the slope created in the seventh step SP 7 is nonlinear, the CPU 21 calculates the fluorescence life for each of linear portions having different gradients and curved portions of the slope shown in the afterglow change table in the eighth step SP 8 .
  • the fluorescence life measuring apparatus 1 irradiates a fluorescent material placed on the rotating disc stage 2 with excitation light and images afterglow of emitted fluorescence caused by the excitation light in the imaging unit 6 . Then, the fluorescence life measuring apparatus 1 detects the elapsed time from a fluorescence position and afterglow strength at a target afterglow position and calculates the fluorescence life from the afterglow strength and elapsed time.
  • the fluorescence life measuring apparatus 1 captures the fluorescence life in the form of an afterglow image in the rotation direction, which allows obtaining of the strength change of light occurring in a short period without using a streak camera, resulting in a simple configuration of the fluorescence life.
  • the fluorescence life measuring apparatus 1 momentarily irradiates the fluorescent material placed on the rotating disc stage 2 with laser light at a predetermined period.
  • the fluorescence life measuring apparatus 1 can be configured more simply than the case of using pulsed laser light.
  • the fluorescence life measuring apparatus 1 can reduce the property change of the fluorescent material caused by the excitation light in comparison with the case of continuously irradiating with pulsed laser light while measuring, which allows correctly measuring of the fluorescence life of the fluorescent material to be correctly measured.
  • the fluorescence life measuring apparatus 1 calculates the fluorescence life for each of linear portions having different gradients and curved portions of those lines.
  • the fluorescence life measuring apparatus 1 can calculate the fluorescence life of the fluorescent material.
  • the fluorescence life measuring apparatus 1 captures the fluorescence life in the form of an afterglow image in the rotation direction, which allows obtaining of the strength change of light occurring in a short period without using a streak camera, resulting in a simple configuration of the fluorescence life.
  • the rotation speed of the disc stage 2 or the angle of view of the imaging unit 6 is adjusted so that the ratio of the strength at the tail position to that at the head position of the imaged afterglow falls within a defined range.
  • the angle of view of the imaging unit 6 may be fixed so that the entire disc stage 2 falls within the imaging range.
  • This configuration allows calculating of the fluorescence life in a way similar to the above embodiment while keeping constant the ratio of the fluorescence strength and the afterglow strength within the imaging range, without the above-described adjustment.
  • the above embodiment in which the rotation speed of the disc stage 2 or the angle of view of the imaging unit 6 is adjusted is preferable.
  • the spindle motor SM is used to rotationally move the stage.
  • the moving means is not limited to one that rotationally moves the stage.
  • one that linearly moves the stage can be used.
  • the moving means has only to be able to provide a constant moving speed in an entire process or a portion thereof.
  • the fluorescent material to be measured is placed on the entire disc stage 2 in the form of a film.
  • the placement position is not limited to the entire stage, and further, the placement may be performed in any other form than the film.
  • the invention can be utilized in bio-based applications, such as gene testing, pharmaceutical origination or patient follow-up.
  • 1 fluorescence life measuring apparatus 2 disc stage, 2 A disc substrate, 2 B reflection film, 2 C fluorescence film, 3 laser light source, 4 optical system, 5 focus control unit, 6 imaging unit, 7 measuring unit, 21 CPU, 22 bus, 23 ROM, 24 RAM, 31 driver, 32 preprocessor, 33 fluorescence life calculator, SM spindle motor, SA rotating shaft

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
US13/143,518 2009-01-16 2010-01-07 Fluorescence life measuring apparatus, fluorescence life measuring method and program Expired - Fee Related US8618506B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2009-007722 2009-01-16
JP2009007722A JP5169857B2 (ja) 2009-01-16 2009-01-16 蛍光寿命測定装置、蛍光寿命測定方法及びプログラム
PCT/JP2010/050364 WO2010082611A1 (fr) 2009-01-16 2010-01-07 Dispositif de mesure de la durée de vie de la fluorescence, procédé de mesure de la durée de vie de la fluorescence, et logiciel

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US8618506B2 true US8618506B2 (en) 2013-12-31

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EP (1) EP2381241B1 (fr)
JP (1) JP5169857B2 (fr)
CN (1) CN102272583B (fr)
WO (1) WO2010082611A1 (fr)

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JP2012132742A (ja) * 2010-12-21 2012-07-12 Fujifilm Corp 時間分解蛍光測定装置、及び方法
CN103592277B (zh) * 2013-11-20 2017-01-11 中国科学技术大学 一种高精度荧光寿命测量装置
CN105300949B (zh) * 2015-11-26 2019-06-11 浙江大学 一种荧光寿命测量方法及装置
CN107860756A (zh) * 2017-11-12 2018-03-30 武汉能斯特科技有限公司 一种测量荧光寿命的方法和装置
CN109100021A (zh) * 2018-02-27 2018-12-28 武汉能斯特科技有限公司 一种时间分辨的光谱和寿命测量模块及装置
CN110632039A (zh) * 2018-06-22 2019-12-31 武汉能斯特科技有限公司 一种高通量测量荧光磷光寿命的转盘和装置
CN112714866A (zh) 2018-09-18 2021-04-27 国立大学法人东京大学 物质确定装置、物质确定方法以及物质确定程序
JP7345939B2 (ja) * 2020-03-17 2023-09-19 国立大学法人 東京大学 状態特定装置、状態特定方法、および状態特定プログラム

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EP2381241A1 (fr) 2011-10-26
EP2381241A4 (fr) 2014-07-23
CN102272583B (zh) 2014-02-26
JP2010164468A (ja) 2010-07-29
JP5169857B2 (ja) 2013-03-27
WO2010082611A1 (fr) 2010-07-22
CN102272583A (zh) 2011-12-07
US20110266458A1 (en) 2011-11-03
EP2381241B1 (fr) 2020-03-18

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